1. Field of the Invention
The present invention relates generally to methods and compositions for monitoring the processing of .beta.-amyloid precursor protein. More particularly, the present invention relates to the use of such methods and compositions for the diagnosis, prognosis, and monitoring response to therapy of Alzheimer's disease, and for screening and evaluation of potential drugs for the treatment of Alzheimer's disease.
Alzheimer's disease is characterized by the presence of numerous amyloid plaques and neurofibrillary tangles (highly insoluble protein aggregates) present in the brains of Alzheimer's disease patients, particularly in those regions involved with memory and cognition. While in the past there was significant scientific debate over whether the plaques and tangles are a cause or are merely the result of Alheimer's disease, recent discoveries indicate that amyloid plaque is a causative precursor or factor. In particular, it has been discovered that the production of .beta.-amyloid peptide, a major constituent of the amyloid plaque, can result from mutations in the gene encoding amyloid precursor protein, a protein which when normally processed will not produce the .beta.-amyloid peptide. The identification of mutations in the amyloid precursor protein gene which cause familial, early onset Alheimer's disease is the strongest evidence that amyloid metabolism is the central event in the pathogenic process underlying the disease. Four reported disease-causing mutations include with respect to the 770 isoform, valine.sup.717 to isoleucine (Goate et al. (1991) Nature 349:704-706), valine.sup.717 to glycine (Chartier Harlan et al. (1991) Nature 353:844-846, valine.sup.717 to phenylalanine (Murrell et al. (1991) Science 254:97-99) and with respect to the 695 isoform, a double mutation changing lysine.sup.595 -methionine.sup.596 to asparagine.sup.595 -leucine.sup.596 (Mullan et al. (1992) Nature Genet 1:345-347). Moreover, .beta.-amyloid peptide is toxic to brain neurons, and neuronal cell death is associated with the disease.
Thus, the ability to monitor cellular processing of the amyloid precursor protein would be of significant value in the diagnosis, prognosis, and therapeutic supervision of Alzheimer's disease. In particular, it would be desirable to identify minimally invasive procedures for screening and evaluating detectable diagnostic markers in readily obtainable patient samples, such as serum, cerebrospinal fluid (CSF), and the like.
A number of potential diagnostic markers for Alzheimer's disease have been proposed. Of particular interest to the present invention are certain fragments of the amyloid precursor protein, including carboxy-terminal fragments (such as the .beta.-amyloid peptide itself and fragments thereof), and amino-terminal fragments (such as certain 25 kD, 105 kD, and 125 kD fragments). As yet, none of the proposed markers has proved to be definitive for the antemortem diagnosis or monitoring of Alzheimer's disease.
Thus, it would be desirable to identify additional and alternative diagnostic markers for Alzheimer's disease. Such markers should be useful by themselves and/or in combination with other diagnostic markers and procedures. Preferably, the diagnostic markers would be detectable in body fluids, such as CSF, blood, plasma, serum, urine, tissue, and the like, so that minimally invasive diagnostic procedures can be utilized.
Of further interest to the present invention are in vitro systems and methods for screening candidate drugs for the ability to inhibit or prevent the production of .beta.-amyloid plaque. It would be desirable to provide methods and systems for screening test compounds for the ability to inhibit or prevent the conversion of amyloid precursor protein to .beta.-amyloid peptide. In particular, it would be desirable to base such methods and systems on metabolic pathways which have been found to be involved in such conversion, where the test compound would be able to interrupt or interfere with the metabolic pathway which leads to conversion. Such methods and systems should be rapid, economical, and suitable for screening large numbers of test compounds.
2. Description of the Background Art
.beta.-amyloid peptide (also referred to as A4, .beta.AP, A.beta., or A.beta.P; see, U.S. Pat. No. 4,666,829 and Glenner and Wong (1984) Biochem. Biophys. Res. Commun. 120:1131-1135) is derived from .beta.-amyloid precursor protein (.beta.APP), which is expressed in differently spliced forms of 695, 751, and 770 amino acids. See, Kang et al. (1987) Nature 325:773-776; Ponte et al. (1988) Nature 331:525-527; and Kitaguchi et al. (1988) Nature 331:530-532. Normal processing of amyloid precursor protein involves proteolytic cleavage at a site between residues Lys.sup.16 and Leu.sup.17 (as numbered for the .nu.AP region where Asp.sup.597 is residue 1 in Kang et al. (1987)), supra, near the transmembrane domain, resulting in the constitutive secretion of an extracellular domain which retains the remaining portion of the .beta.-amyloid peptide sequence (Esch et al. (1990) Science 248:1122-1124). This pathway appears to be widely conserved among species and present in many cell types. See, Weidemann et al. (1989) Cell 57:115-126 and Oltersdorf et al. (1990) J. Biol. Chem. 265:4492-4497. This normal pathway cleaves within the region of the precursor protein which corresponds to the .beta.-amyloid peptide, thus apparently precluding its formation. Another constitutively secreted form of .beta.APP has been noted (Robakis et al. Soc. Neurosci. Oct. 26, 1992, Abstract No. 155, Anaheim, Calif.) which contains more of the .beta.AP sequence carboxy-terminal to that form described by Esch et al. supra.
Golde et al. (1992) Science 255:728-730, prepared a series of deletion mutants of amyloid precursor protein and observed a single cleavage site within the .beta.-amyloid peptide region. Based on this observation, it was postulated that .beta.-amyloid peptide formation does not involve a secretory pathway. Estus et al. (1992) Science 255:726-728, teaches that the two largest carboxy-terminal proteolytic fragments of amyloid precursor protein found in brain cells contain the entire .beta.-amyloid peptide region.
PCT application WO 92/00521 describes methods for evaluating Alzheimer's disease based on measuring the amounts of certain 25 kD, 105 kD, and 125 kD soluble derivatives of amyloid precursor protein in a patient's cerebrospinal fluid. FIG. 3 of WO 92/00521 suggests that cleavage of amyloid precursor protein may occur adjacent to the amino-terminus of .beta.-amyloid peptide to produce a soluble amino-terminal fragment, but no evidence or discussion of such cleavage is presented in the application. Kennedy et al. (1992) Neurodegeneration 1:59-64, present data for a form of secreted .beta.APP, which was characterized by its reactivity with antibodies to residues 527-540 of .beta.APP and the lack of reactivity with antibodies to the first fifteen residues of .beta.AP. No direct evidence is provided to suggest the cleavage site or identity of the carboxy-terminus of the .beta.APP form. PCT application WO 91/16628 describes methods for diagnosing disease based on detection of amyloid precursor proteins and fragments thereof utilizing antibodies to protease nexin-2 or amyloid precursor protein.
Recent reports show that soluble .beta.-amyloid peptide is produced by healthy cells into culture media (Haass et al. (1992) Nature 359:322-325) and in human and animal CSF (Seubert et al. (1992) Nature 359:325-327).